Protein conjugation lies at the heart of the discovery and development of protein therapeutics. Chemical modification strategies typically employ a two step process where the first step involves site-specific modification of the protein and the second step is the installation of an entity of interest. The selectivity of the second step is often a consequence of the chemospecific reactivity of a donor-acceptor pair, e.g., aminooxy donor-carbonyl acceptor, whose reactivity is orthogonal to that of peptide side-chains. (Tam et al., Biopolymers. 51:311-32, 1999)
The first step involving modification of the protein can be difficult to effect site-specifically by chemical methods in view of the presence of many peptide residues of the same type. Accordingly, limited success has been achieved in such site-specific modifications, although enzymes have been used to effect these transformations. For example, it has been demonstrated that group modification agents with minimal binding determinants can sometimes react site-specifically due to enzyme active sites, e.g., active site serines of proteinases (Means et al., Chemical Modification of Proteins, Holden-Day, Inc., San Francisco, 1971.)
For proteins that have not evolved to do such chemistry, the challenges for site-specific labeling are far greater than for the construction of active-site directed reagents. For such proteins the challenges can be likened to the development of site-specific modifications of non-active site residues of enzymes. Thus, other than for active-sites, and allosteric sites that have evolved to bind enzyme modulators, site-specific labeling reagents (affinity labels) are lacking and novel approaches are required to fill that void. Amino acid residues usually have little to distinguish their reactivity from others in the same class, with the exception of cysteine thiols whose chemistry is quite distinct from other peptidic side chain functionality. Alternatively, strategies for the installation of functional groups that can engage in orthogonal conjugative reactions can be useful for the selective modification of proteins.
For these reasons, methods for the site-specific modification and ligation of proteins would be useful for the synthesis of modified peptide, polypeptide, and protein adducts and use of the adducts in radio-labeling, molecular imaging and protein therapeutic applications, and in methods of medical treatment.